Signaling pathways that sense nutrient availability are integral to cellular defense and tissue homeostasis. These pathways have become important drug targets in the treatment of cancer, autoimmune and inflammatory diseases. Our lab has shown that the natural product derivative halofuginone (HF) targets human glutamyl-prolyl- tRNA synthetase (EPRS) and inhibits its proly-tRNA synthetase (PRS) activity to confer therapeutic benefits. AARSs, such as EPRS, are ubiquitous, essential enzymes that fuse an amino acid to its cognate tRNA. HF inhibition of EPRS and environmental depletion of amino acids share a common feature in that each produce cellular accumulation of uncharged tRNAs, and subsequent activation of the AAR pathway. Significantly, we now show that HF treatment selectively inhibits inflammatory responses in diverse cell types and that these therapeutic benefits occur in cells that lack GCN2, the signature effector of the AAR pathway. We further find that other treatments that trigger the accumulation of uncharged tRNAs can mimic key aspects of HF treatment, also in the absence of GCN2. These observations are compelling because GCN2 is thought to be the sole sensor and transducer of the uncharged tRNA signal in mammalian cells. These data strongly suggest both the existence of a novel sensor of uncharged tRNA, and the presence of an unrecognized signaling pathway that responds to amino acid restriction. We further show that GCN1, an upstream regulator of the AAR, is required for these HF-driven tissue benefits, indicating that GCN1 constitutes a branch point from the AAR pathway. We surmise that local amino acid depletion and pharmacologic inhibition of AARSs trigger a novel, therapeutically relevant signaling pathway that branches from the AAR. The premise of this proposal is that hGCN1 is an essential component of this pathway that links amino acid insufficiency to the suppression of pathologic tissue remodeling. We plan to use knowledge of yeast GCN1, and its functional interactors, to predict and identify key mammalian components of this signaling apparatus. Study of this structure/function relationship will help us to better understand how this large scaffold protein senses and transduces a therapeutically important signal. We also will identify transcriptional regulatory elements shared by diverse downstream responses to better understand the links between amino acid deprivation and the programmed suppression of inflammatory tissue response. Completion of this work will establish a new pathway for nutrient sensing in eukaryotic cells and elucidate its link to therapeutic modulation of tissue damage. !